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. 2018 Dec 11;9:5286. doi: 10.1038/s41467-018-07722-9

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — The structural basis of specificity and multispecificity among designed pairs. a Structure models show high polarity in designed, high-specificity interfaces. Im^des1 buries positively charged Lys31 at the interface, which is compensated by negatively charged Glu78 on the cognate colE^des1 and by hydrogen bonding to backbone atoms on Im loop I. colE^des2/Im^des2 interact through a hydrogen-bond network that involves hotspot residue Tyr52. b, c Im Loop I preorganization predicts Im specificity. b Structure models show that stabilizing interactions within loop I of the high-specificity designs preorganize the loop I backbone in the designed conformation. Loop I of Im^des3 is configured by Pro28 and side chain-backbone hydrogen bonds. Loop I of Im^des2 is stabilized by a network of side chain-backbone polar interactions. c Modeling the sequence of the high-specificity Im^des3 on alternative backbone conformations observed in the Protein Data Bank converges on low-energy conformations only in the vicinity of the designed backbone conformation (blue; vertical cyan line at 0.9Å rmsd). Models of the multispecific Im^des7, by contrast, diverge to multiple minima away from the designed conformation (red). Z-scores indicated in parentheses. d Models of the relaxed non-cognate colE/Im pairs suggest that low binding affinity results from cavities, electrostatic repulsion and unsatisfied polar atoms at the interface. The model of colE^wt2/Im^des3.5 (left), with K_D ≥ 100,000 nM, shows a large cavity (yellow) in loop I region and reorientation of the hotspot Phe86 (cognate colE^des3 orientation in gray). Model of colE^wt2/Im^des1 (right), with K_D ≥ 100,000 nM, shows positively charged Lys on colE^wt2 that faces the positively charged loop I region on Im^des1 (surface colored according to the electrostatic potential), and a negatively charged Glu that faces a surface of the same charge on helix II of Im^des1. The computed binding energies for these non-cognate interactions are poor (−12.4 Rosetta energy units [R.e.u] for colE^wt2/Im^des3.5 and −15.8 R.e.u for colE^wt2/Im^des1) compared to tight binding of the cognate colE^des3.5/Im^des3.5, colE^des1/Im^des1, and colE^wt2/Im^wt2 counterparts (−36.0, −39.9, and −34.6 R.e.u, respectively)